The present invention relates to 2-(1H-indol-3-yl)-2-oxo-acetamides having antitumor activity, particularly against solid tumors, more precisely colon and lung tumors.
Colo-rectal carcinoma is one of the most common tumors in Western countries as it accounts for about 421,000 new cases each year in the world, and it is the most frequent cause of death, except lung and breast cancers.
Surgical cure is possible in about 40-50% of patients, the remaining patients can be treated with combined chemotherapy, to obtain complete remission in a percentage not higher than 5%.
Colo-rectal tumors are usually refractory or poorly sensitive to the presently available chemotherapy, and the only agent who has some efficacy for this type of cancer is 5-fluorouracil.
No therapeutical alternatives are at present available in case of failure of the combination chemotherapy based on 5-FU. There is therefore strong need for novel medicaments active against this type of tumors.
WO 99712917 in the name of Roche Diagnostics discloses 4-ureido and thioureido 2(5H)-furanone or 2(5H)-thiophenone derivatives with antitumor activity, particularly against colon tumors.
WO 98/09946 in the name of Asta Medica discloses indol-3-glyoxylamide derivatives. The compounds are substituted at the amido nitrogen with aromatic and pyridyl residues and are reported to have antiasthmatic, antiallergic, immunosuppressive and immunomodulating activities.
In Proceedings of the American Association for Cancer Research, volume 40, abstract 1893 and 4110, 1999, the compound N-(4-pyridyl)-2-(1-(4-chlorobenzyl)-1H-indol-3-yl)-glyoxylamide (D-24,851) is described to have in vitro and in vivo antitumor effects.
It has now been found that N-(5-oxo-2,5-dihydrofuran-3-yl) or N-(5-oxo-2,5-dihydrothiophen-3-yl)-2-(1H-indol-3-yl)-2-oxo-acetamido derivatives have marked antitumor activity, particularly against human solid tumors.
The compounds of the invention can be represented by the general formula (I): 
wherein:
R1, R2 and R5 are independently hydrogen or a C1-C6 alkyl group;
R3 is hydrogen, C1-C4 alkyl, aralkyl, optionally substituted phenyl;
R4 is hydrogen, straight or branched C1-C8 alkyl, C5-C6 cycloalkyl; aralkyl; heteroaralkyl;
X is one or more groups, at most four, independently selected from hydrogen; C1-C6 alkyl; C1-C6 haloalkyl; C1-C6 hydroxyalkyl; C1-C6-aminoalkyl; C1-C6-alkoxy-C1-C6-alkyl; C1-C18-acyloxy-C1-C6-alkyl; hydroxy; C1-C4 alkoxy; C1-C3 haloalkoxy; phenoxy; aralkoxy; C1-C3 acyloxy; amino; C1-C3 alkylamino; C1-C3-acylamino; C1-C3-alkylsulfonylamino; aroylamino; halogen; nitro; cyano; trifluoromethyl; carboxy; C1-C6 alkoxycarbonyl; a RaRbN(CH2)nC(xe2x95x90O)xe2x80x94 group wherein Ra and Rb are independently hydrogen, C1-C3-alkyl or Ra and Ro together with the nitrogen atom they are linked to form a pyrrolidino, piperidino, piperazino or morpholino ring and n=0 or an integer from 1 to 4; a RaRcN(CH2)nC(xe2x95x90O)xe2x80x94 group wherein Ra and n are as above defined and Rc is a C1-C4-alkoxycarbonyl group; a R1C(xe2x95x90O)xe2x80x94 group wherein R1 is as above defined; sulfonyl; mercapto; C1-C4-alkylthio; C1-C4-alkylsulfinyl; C1-C4-alkylsulfonyl; aminosulfonyl; C1-C3-alkylamiosulfonyl; a group xe2x80x94P(xe2x95x90O)(OR1)(OR2) being R1 and R2 as above defined; a group (E)xe2x80x94 or (Z)xe2x80x94C(R1)xe2x95x90C(R2)xe2x80x94C(xe2x95x90O)R6 wherein R6 is hydroxy, C1-C6-alkoxy, NRaRb or a group of formula RaRbN(CH2)mNR1xe2x80x94, being m an integer from 2 to 4 and R1, R2, Ra, and Rb as above defined;
Y is an oxygen or sulfur atom, and the isomers, enantiomers and mixtures thereof.
The invention also relates to the salts of compounds of formula (I) obtainable by reacting non toxic acids or bases with the ionisable groups present in compounds (I).
Optionally substituted phenyl preferably means phenyl, 4-methylphenyl, 2,4-dimethoxy-phenyl, 4-methoxy-phenyl, 4-nitro-phenyl, 3-chlorophenyl, 4-hydroxyphenyl, 3,5-dimethoxy-4-hydroxy-phenyl, 3-cyano-phenyl, 2-hydroxyphenyl, 2-carboxyphenyl.
Aralkyl preferably means benzyl, phenethyl, naphthylmethyl, biphenylmethyl, optionally substituted with one or more chloro, fluoro, trifluoromethyl, nitro, cyano, methylsulfonyl, tert-butyl groups.
Heteroaralkyl preferably means pyridylmethyl.
R1 and R2 and R3 and R5 are preferably hydrogen and methyl.
R4 is preferably hydrogen; methyl; benzyl substituted on the benzene ring with one or more groups selected from methyl, t-butyl, fluorine, chlorine, bromine, hydroxy, acetoxy, methoxy, trifluoromethoxy, benzyloxy, trifluoromethyl, cyano, nitro, amino, acetylamino, methylsulfonylamino, methylmercapto, methylsulfinyl, methylsulfonyl, phenyl, ethoxycarbonyl, carboxy, carboxymethyl, (ethoxycarbonyl)methyl, (tert-butoxycarbonyl)methyl, (benzyloxycarbonyl)methyl, (dimethylcarbamoyl)methyl; xcex1-naphthyl, xcex2-naphthyl; 4-pyridyl; 4-pyridyl-N oxide.
X is preferably methyl, ethyl, fluorine, chlorine, bromine, hydroxy, acetoxy, methoxy, phenoxy, trifluoromethoxy, trifluoromethyl, cyano, nitro, amino, acetylamino, methylsulfonylamino, methylmercapto, methylsulfinyl, methylsulfonyl, carboxy, methoxycarbonyl, tert-butoxycarbonyl, diethylcarbamoyl, (2-aminoethyl)carbamoyl, (2-dimethylaminoethyl)carbamoyl, (E)- and (Z)-2-carboxyethen-1-yl, (E)- and (Z)-(2-tert-butoxycarbonyl)ethen-1-yl, (E)- and (Z)xe2x80x94(ethoxycarbonyl)ethenyl, hydroxymethyl, and allyloxymethyl.
Y is preferably an oxygen atom.
The compounds of the invention can be prepared by reacting compounds of formula (II) 
wherein R3, and R4 and X are as defined above,
with a compound of formula (HI) 
wherein Y, R1 and R2 are as defined above.
The reaction is carried out in a solvent such as ethyl ether, isopropyl ether, methyl-tert-butyl ether, tetrahydrofuran, dioxane, 1,2-dimethoxyethane, dichloromethane, 1,2-dichloroethane, toluene, dimethylformamide, dimethylacetamide, dimethylsulfoxide, at a temperature ranging from 0xc2x0 C. to the reflux temperature of the solvent, and using 1 to 3 molar equivalents of compounds of formula (III). Optionally, the reaction can be performed in the presence of the carbonate of an alkaline- or alkaline-earth metal.
The reaction is preferably carried out in an ether solvent such as ethyl ether, THF, or 1,2-dimethoxyethane, at a temperature ranging from room temperature to 80xc2x0 C., in the presence of at least one equivalent of potassium carbonate.
The resulting compounds of formula (I) can subsequently be transformed into other compounds of formula (I) according to the procedures conventionally used for the transformation of functional groups, for example reactions such as hydrolysis of ester groups, esterification of carboxylic acids, amidation, and the like. For example, when in compounds of formula (II) X and R4 contain substituents which interfere with the reaction of compounds of formula (II) with compounds of formula (III), suitable protective groups will be used and subsequently removed according to conventional methods.
The compounds of formula I in which R5 is a C1-C6 alkyl group are obtained by alkylation of the compounds of formula I in which R5 is hydrogen with a R5-Hal derivative, wherein Hal is preferably chlorine, bromine or iodine, in the presence of the hydride of an alkali- or alkaline-earth metal.
Compounds of formula (II) are obtained by reacting compounds of formula (IV) 
wherein X, R3 and R4 are as defined above, with oxalyl chloride.
The reaction is usually carried out in a solvent such as ethyl ether, isopropyl ether, tert-butyl methyl ether, tetrahydrofuran, dioxane, dichloromethane, at a temperature ranging from xe2x88x9210xc2x0 C. to 25xc2x0 C. and using from one to two molar equivalents of oxalyl chloride, preferably at 0xc2x0 C. to 25xc2x0 C. in ethyl ether or in tetrahydrofuran and using a slight excess (1.2 molar equivalents) of oxalyl chloride. The reaction is usually completed in 3 hours.
Compounds of formula (IV) are obtained by reacting indoles of formula (V) 
with halides of formula R4-Hal (VI), wherein Hal is preferably chlorine, bromine or iodine, in the presence of acid-binding agents.
The reaction is usually carried out using an equimolar amount or a slight excess of the halide (VI), in a protic, dipolar aprotic or apolar solvent such as ethanol, isopropanol, tert-butanol, tetrahydrofuran, dioxane, dimethylsulfoxide, dimethylformamide, dimethylacetamide, N-methylpyrrolidinone, acetonitrile, in the presence of an alkali or alkaline-earth metal hydroxide or alkoxide or hydride such as sodium hydroxide, sodium hydride, potassium tert-butoxide. The reaction is carried out at a temperature usually ranging from 0xc2x0 C. to the reflux temperature of the solvent, for a time from 30xe2x80x2 to 24 hours, preferably in dimethylsulfoxide in the presence of sodium hydride in equimolar amount to compounds (V), reacting compounds (V) with sodium hydride at 0xc2x0-25xc2x0 C., then adding compounds (VI) and heating to 50-70xc2x0 C. The reaction is usually completed in three hours.
Compounds of formulae (V) and (VI) are known or can be prepared by known methods, and many of them are commercially available.
Compounds of formula (III) wherein Y is oxygen are obtained through the reactions described in the following Scheme starting from known compounds of formula (VII). Compounds (VII) are first converted into enamines (VIII) by reaction with an ammonium salt, such as ammonium acetate. Enamines are subsequently converted into compounds (III) in which Yxe2x95x90O by heating in dimethylformamide. 
Alternatively, compounds (III) in which Y is oxygen or sulfur can be prepared from the known furanediones or thiophenediones (IX) by melting with an ammonium salt such as ammonium acetate. 
The compounds according to the invention have been pharmacologically tested against four human tumor cell lines: HT 29 (colon carcinoma), PC 3 (prostate carcinoma), H 460M (lung carcinoma), MKN-45 (gastric carcinoma). Cells were incubated with the tested compound for 144 hours, then cytotoxicity was determined by using the MTT assay (Mosman, T. xe2x80x9cRapid Colorimetric Assay for Cellular Growth and Survival: Application to Proliferation and Cytotoxicity Assayxe2x80x9d; J. Immunolog. Methods, (1983), 65, 66; Green, L. M., xe2x80x9cRapid Colorimetric Assay for Cell Viability; Application to the Quantitation of Cytotoxic and Growth Inhibitory Lymphokinesxe2x80x9d, J. Immunol. Methods, (1984),;70, 257-268).
The obtained data evidenced that the compounds according to the present invention have remarkable activity against solid tumors, in particular colon and lung tumors.
The compounds of the invention can be administered in doses ranging from 0.01 mg to 1 g/kg body weight daily. A preferred dosage regimen may range from about 1 mg to about 500 mg/kg body weight daily, using such unitary doses as to administer in 24 hours from about 70 mg to about 3.5 g of the active substance to a patient weighing about 70 Kg. Such dosage regimen may be adjusted in order to obtain a better therapeutical effect. For example, dosages may be adjusted in consideration of the therapeutical conditions of the patient. The active compounds of the invention can be administered through the oral, intravenous, intramuscular or subcutaneous route.
The compounds of the invention may be administered, according to well-known therapeutical procedures, in combination with other agents used to induce the regression of tumors, in order to synergistically increase the antitumor effects of said compounds. Examples of compounds which can be used in combination with the compounds of the invention are cisplatin, carboplatin, doxorubicin, topotecan, taxol, taxotere, vincristine, 5-fluorouracil.
The pharmaceutical compositions according to the present invention contain therapeutically effective amounts of at least one compound of the invention in mixture with pharmaceutically acceptable excipients.
The oral compositions will generally include an inert diluent or an edible carrier and may be included in gelatin capsules or compressed into tablets. Other forms suitable for oral administration are capsules, pills, elixirs, suspensions or syrups.
The tablets, pills, capsules and similar compositions may contain the following ingredients (in addition to the active substance): a binder such as a microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose; a disintegrating agent such as alginic acid, primogel, corn starch and the like; a lubricant such as magnesium stearate; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharine or a flavoring agent such as peppermint, methyl salicylate or orange flavor. When the chosen composition is in form of capsules, it may contain in addition a liquid carrier such as a fatty oil. Other compositions may contain other various materials which modify the physical form, such as coating agents (for tablets and pills) such as sugar or shellac. The materials used in the preparation of the compositions should be pharmaceutically pure and not toxic at the employed dosages.
For the preparation of pharmaceutical compositions for the parenteral administration, the active ingredient may be incorporated into solutions or suspensions, which may include in addition the following components: a sterile diluent such as water for injection, saline solution, oils, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol; antioxidants such as ascorbic acid or sodium bisulphite; chelating agents such as ethylenediaminotetraacetic acid; buffers such as acetates, citrates or phosphates and agents for adjusting the solution tonicity such as sodium chloride or dextrose. The parenteral preparation may be included in ampoules, disposable syringes or glass or plastic vials.